Bioactive proteins are produced in various host cells, ranging from bacteria and yeast to mammalian cells. Mammalian cells as host cell are preferred when the protein requires certain posttranslational modifications, such as glycosylation to function properly. In general, proteins produced in mammalian cells are expressed from a so-called ‘transgene’ encoding the protein of interest. To ensure that the right, protein-producing cell is selected, the transgene coding for the gene of interest is coupled to a second transgene encoding a selectable marker that most often is placed on the same vector. A common problem is that the stringency of selection is often low, meaning that the cell has to make only very small amounts of selection protein in order to survive the toxic selective conditions. If only a limited expression of the selectable marker protein is required for selection of the cells, this also has implications for the expression levels of the transgenic protein. Low expression levels of selectable marker protein will usually be accompanied by low expression of the transgene protein. This is obviously an unwanted side effect of low selection stringency.
An improvement in selection stringency is seen with the Zeocin selection marker. This is because the Zeocin selection protein does not act as an enzyme but rather stoichiometrically binds two Zeocin selection molecules without further processing them. As a consequence, the cell must produce much more molecules of a stoichiometric selectable marker such the Zeocin selection protein as compared to an enzymatic selectable marker protein of which a single molecule is capable of katalysing inactivation many molecules of the selection agent. When coupled to a gene of interest, the higher stringency of the stoichiometric selectable marker usually results in higher levels of mRNA and/or expression of the gene product of interest.
Because stably transfected clones can only be selected for the expression levels of the selection marker and not for the expression level of the gene of interest, it is preferred that the expression of the gene of interest is directly linked to the expression level of the selection marker. One way of achieving this by placing an IRES (Internal Ribosome Entry Site) sequence between the gene of interest and the gene encoding the selection marker. This creates a single bicistronic mRNA from which both the gene product of interest and the selection protein are translated (Rees et al., 1996, Biotechniques 20: 102-110). A high level of expression of the selectable marker, e.g. by using a high stringency marker, is thereby directly coupled to a high level of expression of the gene product of interest. This is an accepted and often employed method procedure for selection of clones that express relatively high levels of the gene product of interest (see e.g. WO 03/106684, WO 2006/005718 and WO 2007/096399).
The stringency of selection can be further increased by using selectable markers that harbor mutations that attenuate but do not completely destroy the activity of the selection marker. Under the same selective conditions, higher levels of the impaired selection protein will be required as compared to the wild type selection protein. When coupled to the gene of interest through an IRES sequence, the higher mRNA levels of the impaired selection marker warrant that there will also be more mRNA of the gene of interest available for translation. (see e.g. WO 01/32901 and WO 2006/048459)
In another example of high selection stringency systems the translation initiation of the selection marker protein is severely impaired by using sub-optimal, non-ATG codons for initiation of translation of the selectable marker protein. These selection systems have been termed STAR-Select (Otte et al. (2007) Biotechnol. Progr. 23(4):801-807; WO 2006/048459 and WO 2007/096399).
Recently, the present inventors developed a novel stringent selection principle whereby translation initiation of the selection marker protein is severely impaired by placing a coding sequence for a short peptide immediately upstream of a selection marker, thereby requiring the ribosome to re-initiate translation at the translation initiation codon of the selectable marker protein (co-pending application PCT/NL2010/050367). In this system, the stringency of selection can be fined tuned by increasing the length of the short peptide: when the short peptide becomes longer, the translation machinery will have increasing difficulties to re-initiate at the translation initiation codon of the selectable marker protein. In combination with the Zeocin selectable marker protein this stringent selection system has been dubbed the “ppZeo selection system” (pp=petite peptides).
However, one problem with the high-stringency selection systems is that the number of colonies obtained after transformation is significantly reduced, even down to a level that hardly any colonies are obtained. This problem has been addressed by the inclusion in expression vectors of expression enhancing sequences such as Locus Control Regions (LCR; Needham et al., 1995. Protein Expr Purif 6:124-131) or STARs (WO 03/004704; WO 03/106674; WO 03/106684; WO 2006/005718; WO 2006/048459 and WO 2007/096399). WO 2006/123097 discloses that also DNA fragments from the promoter region of the genes coding for the ribosomal proteins S3 and S11 (RPS3 and RPS11, respectively), when linked to an expression cassette comprising an heterologous promoter, are capable of increasing transcription from the heterologous promoter in the cassette.
There is however, still a need in the art for improved means and methods for high stringency selection of mammalian cells to achieve high production of colonies and/or high expression levels of gene products of interest. In particular, there is still a need for further improved DNA fragments that are capable of increasing expression of expression cassettes comprising highly stringent selectable markers.